Critical to the development and maintenance of multicellular organisms is the regulation of intercellular communication. The primary contacts made between cells and the surrounding environment, including contacts with other cells, are mediated by surface and transmembrane proteins that can transduce signals to the interior of the cell modulating biochemical pathways. Cadherins, one class of transmembrane glycoproteins, are characterized by a long extracellular domain which interacts with cadherins on the opposite membrane of adhering cells and a cytoplasmic domain anchored to the actin cytoskeleton through another class of proteins, the catenins. Because cell adhesiveness is reduced during metastasis, much attention has been focused on the cell adhesion system and, in particular, the role of the cadherins and catenins in this process.
Beta catenin (also known as cadherin-associated protein and .beta.-Catenin), a member of the catenin family of cytosolic proteins and key mediator of the Wnt signaling pathway, was originally isolated because of its association with the cytoplasmic domain of E-cadherin, an epithelial-specific cell-adhesion molecule (Willert and Nusse, Curr. Opin. Genet. Dev., 1998, 8, 95-102). It was demonstrated that Beta catenin undergoes phosphorylation upon growth factor stimulation resulting in reduced cell adhesion, thereby functioning as a component of adherin junctions which are multiprotein complexes that mediate cell adhesion, cell--cell communication and cytoskeletal anchoring. Since then it has been demonstrated that Beta catenin performs a dual role in the cell, in that free pools of Beta catenin mediate signal transduction pathways involving T-cell factor/lymphoid-enhancer factor (TCF/LEF) transcription factor complexes (Korinek et al., Science, 1997, 275, 1784-1787; Peifer, Science, 1993, 262, 1667-1668).
The role of Beta catenin in the development of several types of cancer, more specifically colorectal cancer and melanomas, has been investigated and shown to be regulated by the expression product of the APC (adenomatous polyposis of the colon) gene (Korinek et al., Science, 1997, 275, 1784-1787; Morin et al., Science, 1997, 275, 1787-1790). The APC protein normally binds Beta catenin in conjunction with TCF/LEF forming a transcription factor complex. It is believed that APC regulates the concentration of Beta catenin in the cytosol by controlling its degradation. In studies of the relationship between Beta catenin and APC, Ilyas et al. found four different mutations in Beta catenin including deletions and missense mutations in 21 colorectal cancer cell lines. The full-length Beta catenin protein was detected in all cell lines studied, but failed to show complex formation with APC in some cell lines (Ilyas and Tomlinson, J. Pathol., 1997, 182, 128-137). In other studies, Rubinfeld et al. detected abnormally high amounts of Beta catenin in 7 of 26 human melanoma cell lines and, in 6 of these 7, unusual splicing (deletions of exons 2 and 3 or 2,3 and 4) or missense mutations (TCT to TTT mutations causing a serine 45 to phenylalanine 37 mutation in 3 of 4 cases and a TCT to TAT mutation causing a serine 45 to tyrosine 45 mutation in another case) in Beta catenin; with APC being missing or altered in two of the six (Rubinfeld et al., Science, 1997, 275, 1790-1792). Finally Morin et al. demonstrated that mutations of Beta catenin that altered phosphorylation sites rendered the cells insensitive to APC-mediated downregulation of Beta catenin and that this disrupted mechanism was critical to colorectal tumorigenesis (Morin et al., Science, 1997, 275, 1787-1790).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of Beta catenin and strategies aimed at inhibiting Beta catenin function have involved the use of antibodies, antisense oligonucleotides, chemical inhibitors and gene knock-outs in mice. Furthermore, most of these studies have been focused on elucidating the role of Beta catenin in the process of embryogenesis.
Although Beta catenin is expressed ubiquitously, it is specifically required in ectodermal cell layer development. In knock-out mice lacking Beta catenin, cells detached from the ectodermal cell layer and were dispersed into the proamniotic cavity and no mesoderm formation was observed (Haegel et al., Development, 1995, 121, 3529-3537).
Studies involving an antisense Beta catenin 18-mer phosphorothioate/phosphodiester oligodeoxynucleotide injected into Xenopus embryos demonstrated that either overexpression of cadherins or underexpression of Beta catenin, through treatment with antisense, was sufficient to inhibit dorsal mesoderm induction indicating an important role for Beta catenin in embryogenesis (Heasman et al., Cell, 1994, 79, 791-803).
Antisense mediated inhibition of Beta catenin was also used as a tool to investigate the effect of Beta catenin in the process of invasive motility in normal and uveal melanomas. In these studies, treatment of cells with a single 20-mer phosphorothioate antisense oligodeoxynucleotide targeting both human Beta catenin and human plakoglobin reduced the invasive motility of both normal and neoplastic mesenchymal cells (Kim et al., Exp. Cell. Res., 1998, 245, 79-90). Additional oligonucleotides targeted to either Beta catenin or plakoglobin (targeted to unique sequences) did not have a significant effect.
These antisense strategies are untested as therapeutic protocols and consequently there remains a long felt need for additional agents capable of effectively inhibiting Beta catenin function.
Antisense technology is, however, emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of Beta catenin expression.